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1.
Biochemistry ; 58(24): 2750-2759, 2019 06 18.
Article in English | MEDLINE | ID: mdl-31117388

ABSTRACT

Aggregation can be a major challenge in the development of antibody-based pharmaceuticals as it can compromise the quality of the product during bioprocessing, formulation, and drug administration. To avoid aggregation, developability assessment is often run in parallel with functional optimization in the early screening phases to flag and deselect problematic molecules. As developability assessment can be demanding with regard to time and resources, there is a high focus on the development of molecule design strategies for engineering molecules with a high developability potential. Previously, Dudgeon et al. [(2012) Proc. Natl. Acad. Sci. U. S. A. 109, 10879-10884] demonstrated how Asp substitutions at specific positions in human variable domains and single-chain variable fragments could decrease the aggregation propensity. Here, we have investigated whether these Asp substitutions would improve the developability potential of a murine antigen binding fragment (Fab). A full combinatorial library consisting of 393 Fab variants with single, double, and triple Asp substitutions was first screened in silico with Rosetta; thereafter, 26 variants with the highest predicted thermodynamic stability were selected for production. All variants were subjected to a set of developability studies. Interestingly, most variants had thermodynamic stability on par with or improved relative to that of the wild type. Twenty-five of the variants exhibited improved nonspecificity. Half of the variants exhibited improved aggregation resistance. Strikingly, while we observed remarkable improvement in the developability potential, the Asp substitutions had no substantial effect on the antigenic binding affinity. Altogether, by combining the insertion of negative charges and the in silico screen based on computational models, we were able to improve the developability of the Fab rapidly.


Subject(s)
Aspartic Acid/chemistry , Immunoglobulin Fab Fragments/chemistry , Amino Acid Substitution , Animals , Antigens/immunology , Computer Simulation , HEK293 Cells , Humans , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/immunology , Mice , Peptide Library , Protein Multimerization/genetics , Protein Stability
2.
Blood ; 130(17): 1903-1910, 2017 10 26.
Article in English | MEDLINE | ID: mdl-28819011

ABSTRACT

Despite recent advances in lymphoma treatment, mantle cell lymphoma (MCL) remains incurable, and we are still unable to identify patients who will not benefit from the current standard of care. Here, we explore the prognostic value of recurrent genetic aberrations in diagnostic bone marrow (BM) specimens from 183 younger patients with MCL from the Nordic MCL2 and MCL3 trials, which represent current standard-of-care regimens. In the univariate model, mutations of TP53 (11%) and NOTCH1 (4%), and deletions of TP53 (16%) and CDKN2A (20%), were significantly associated with inferior outcomes (together with MIPI, MIPI-c, blastoid morphology, and Ki67 > 30%); however, in multivariate analyses, only TP53 mutations (HR, 6.2; P < .0001) retained prognostic impact for overall survival (OS), whereas TP53 mutations (HR, 6.9; P < .0001) and MIPI-c high-risk (HR, 2.6; P = .003) had independent prognostic impact on time to relapse. TP53-mutated cases had a dismal outcome, with a median OS of 1.8 years, and 50% relapsed at 1.0 years, compared to a median OS of 12.7 years for TP53-unmutated cases (P < .0001). TP53 mutations were significantly associated with Ki67 > 30%, blastoid morphology, MIPI high-risk, and inferior responses to both induction- and high-dose chemotherapy. In conclusion, we show that TP53 mutations identify a phenotypically distinct and highly aggressive form of MCL with poor or no response to regimens including cytarabine, rituximab, and autologous stem-cell transplant (ASCT). We suggest patients with MCL should be stratified according to TP53 status, and that patients with TP53 mutations should be considered for experimental frontline trials exploring novel agents.


Subject(s)
Immunotherapy , Lymphoma, Mantle-Cell/drug therapy , Lymphoma, Mantle-Cell/genetics , Mutation/genetics , Tumor Suppressor Protein p53/genetics , Adult , Aged , Bone Marrow/pathology , Cohort Studies , Disease-Free Survival , Female , Humans , Kaplan-Meier Estimate , Lymphoma, Mantle-Cell/pathology , Male , Middle Aged , Multivariate Analysis , Prognosis , Proportional Hazards Models
3.
Blood ; 125(17): 2669-77, 2015 Apr 23.
Article in English | MEDLINE | ID: mdl-25736311

ABSTRACT

Recent studies show that mantle cell lymphoma (MCL) express aberrant microRNA (miRNA) profiles; however, the clinical effect of miRNA expression has not previously been examined and validated in large prospective homogenously treated cohorts. We performed genome-wide miRNA microarray profiling of 74 diagnostic MCL samples from the Nordic MCL2 trial (screening cohort). Prognostic miRNAs were validated in diagnostic MCL samples from 94 patients of the independent Nordic MCL3 trial (validation cohort). Three miRNAs (miR-18b, miR-92a, and miR-378d) were significantly differentially expressed in patients who died of MCL in both cohorts. MiR-18b was superior to miR-92a and miR-378d in predicting high risk. Thus, we generated a new biological MCL International Prognostic Index (MIPI-B)-miR prognosticator, combining expression levels of miR-18b with MIPI-B data. Compared to the MIPI-B, this prognosticator improved identification of high-risk patients with regard to cause-specific, overall, and progression-free survival. Transfection of 2 MCL cell lines with miR-18b decreased their proliferation rate without inducing apoptosis, suggesting that miR-18b may render MCL cells resistant to chemotherapy by decelerating cell proliferation. We conclude that overexpression of miR-18b identifies patients with poor prognosis in 2 large prospective MCL cohorts and adds prognostic information to the MIPI-B. MiR-18b may reduce the proliferation rate of MCL cells as a mechanism of chemoresistance.


Subject(s)
Lymphoma, Mantle-Cell/diagnosis , Lymphoma, Mantle-Cell/genetics , MicroRNAs/genetics , Up-Regulation , Aged , Apoptosis , Biomarkers, Tumor/genetics , Cell Line, Tumor , Cell Proliferation , Disease-Free Survival , Female , Gene Expression Regulation, Neoplastic , Humans , Male , Middle Aged , Prognosis , Prospective Studies , Transfection
4.
Trends Hear ; 28: 23312165241246616, 2024.
Article in English | MEDLINE | ID: mdl-38656770

ABSTRACT

Negativity bias is a cognitive bias that results in negative events being perceptually more salient than positive ones. For hearing care, this means that hearing aid benefits can potentially be overshadowed by adverse experiences. Research has shown that sustaining focus on positive experiences has the potential to mitigate negativity bias. The purpose of the current study was to investigate whether a positive focus (PF) intervention can improve speech-in-noise abilities for experienced hearing aid users. Thirty participants were randomly allocated to a control or PF group (N = 2 × 15). Prior to hearing aid fitting, all participants filled out the short form of the Speech, Spatial and Qualities of Hearing scale (SSQ12) based on their own hearing aids. At the first visit, they were fitted with study hearing aids, and speech-in-noise testing was performed. Both groups then wore the study hearing aids for two weeks and sent daily text messages reporting hours of hearing aid use to an experimenter. In addition, the PF group was instructed to focus on positive listening experiences and to also report them in the daily text messages. After the 2-week trial, all participants filled out the SSQ12 questionnaire based on the study hearing aids and completed the speech-in-noise testing again. Speech-in-noise performance and SSQ12 Qualities score were improved for the PF group but not for the control group. This finding indicates that the PF intervention can improve subjective and objective hearing aid benefits.


Subject(s)
Correction of Hearing Impairment , Hearing Aids , Noise , Persons With Hearing Impairments , Speech Intelligibility , Speech Perception , Humans , Male , Female , Aged , Noise/adverse effects , Middle Aged , Correction of Hearing Impairment/instrumentation , Persons With Hearing Impairments/rehabilitation , Persons With Hearing Impairments/psychology , Perceptual Masking , Hearing Loss/rehabilitation , Hearing Loss/psychology , Hearing Loss/diagnosis , Audiometry, Speech , Surveys and Questionnaires , Aged, 80 and over , Time Factors , Acoustic Stimulation , Hearing , Treatment Outcome
5.
Animals (Basel) ; 14(11)2024 May 30.
Article in English | MEDLINE | ID: mdl-38891673

ABSTRACT

Osteoarthritis is a common degenerative disease in dogs, often manifested as pain, joint swelling, and lameness. Despite the lack of scientific evidence for its treatment efficacy, transcutaneous electrical nerve stimulation (TENS) is used in dogs as a pain-relieving treatment. This randomised single-blinded cross-over study investigated the effect of TENS on gait parameters in fifteen dogs with osteoarthritis. Stance time, swing time, stride time, stride length, peak vertical force (%BW), vertical impulse (%BW*sec), and symmetry indices were obtained using a pressure-sensitive mat. TENS treatment of 80 Hz and 100 µs with an individually selected amplitude was conducted for 45 min once daily for a treatment period of seven or ten days. No significant differences were seen between TENS and placebo for any of the gait parameters. Hence, in this study, TENS did not affect gait parameters, compared to placebo. Further studies are needed to confirm the observations.

6.
Haematologica ; 98(12): 1912-20, 2013 Dec.
Article in English | MEDLINE | ID: mdl-23831920

ABSTRACT

The discovery that the Ten-Eleven Translocation (TET) hydroxylases cause DNA demethylation has fundamentally changed the notion of how DNA methylation is regulated. Clonal analysis of the hematopoetic stem cell compartment suggests that TET2 mutations can be early events in hematologic cancers and recent investigations have shown TET2 mutations in diffuse large B-cell lymphoma. However, the detection rates and the types of TET2 mutations vary, and the relation to global methylation patterns has not been investigated. Here, we show TET2 mutations in 12 of 100 diffuse large B-cell lymphomas with 7% carrying loss-of-function and 5% carrying missense mutations. Genome-wide methylation profiling using 450K Illumina arrays identified 315 differentially methylated genes between TET2 mutated and TET2 wild-type cases. TET2 mutations are primarily associated with hypermethylation within CpG islands (70%; P<0.0001), and at CpG-rich promoters (60%; P<0.0001) of genes involved in hematopoietic differentiation and cellular development. Hypermethylated loci in TET2 mutated samples overlap with the bivalent (H3K27me3/H3K4me3) silencing mark in human embryonic stem cells (P=1.5×10(-30)). Surprisingly, gene expression profiling showed that only 11% of the hypermethylated genes were down-regulated, among which there were several genes previously suggested to be tumor suppressors. A meta-analysis suggested that the 35 hypermethylated and down-regulated genes are associated with the activated B-cell-like type of diffuse large B-cell lymphoma in other studies. In conclusion, our data suggest that TET2 mutations may cause aberrant methylation mainly of genes involved in hematopoietic development, which are silenced but poised for activation in human embryonic stem cells.


Subject(s)
DNA Methylation/genetics , DNA-Binding Proteins/genetics , Gene Expression Profiling/methods , Genome-Wide Association Study/methods , Lymphoma, Large B-Cell, Diffuse/genetics , Mutation/genetics , Proto-Oncogene Proteins/genetics , Aged , Dioxygenases , Female , Humans , Lymphoma, Large B-Cell, Diffuse/diagnosis , Male , Middle Aged
7.
Animals (Basel) ; 11(8)2021 Aug 05.
Article in English | MEDLINE | ID: mdl-34438777

ABSTRACT

Equine dentistry has developed immensely and human dental equipment, such as handpieces, are often used. Measures to avoid the spread of infectious microorganisms are important, but this is challenging since handpieces are difficult to decontaminate. Thus, it is necessary to develop effective IPC measures in equine dentistry. The aim of this study was to contribute to the evidence needed for future evidence-based guidelines on IPC by investigating hygiene in equine dentistry. Used handpieces and dummies (i.e., handpieces not used during dental procedure, reflecting environmental bacterial contamination) and the head support were sampled each day before the first patient, for each patient after treatment, and after decontamination. All equipment was sampled with 3M TM Swab Samplers and the head support additionally sampled with dip slides. After dental procedures, the detected bacterial load was often high on used handpieces, dummies, and the head support. After decontamination, handpieces did not meet the criteria for high-level disinfected equipment. In all but one case decontamination of the head support resulted in a lowered bacterial load. There is a great need for evidence-based guidelines on hygiene in equine dentistry, including IPC measures, to decrease the risk of spreading infectious microorganisms between patients, facilities, and stables.

8.
FEBS Open Bio ; 10(4): 507-514, 2020 04.
Article in English | MEDLINE | ID: mdl-31950675

ABSTRACT

In basic and applied biotechnology, design of affinity ligands has become essential for high-capacity applications such as affinity-based downstream processes for therapeutic molecules. Here, we established a proof-of-concept for the use of multimeric fusion single-chain variable fragment (scFvs) as high-capacity ligands in affinity adsorbents. Mono- and di/tri-scFvs separated by Pro-rich negatively charged linkers were designed, produced, and immobilized to 6% cross-linked agarose beads. Frontal binding experiments with a target protein of 50 kDa resulted in up to 20 mg·mL-1 and 82% in dynamic binding capacity and utilization yield, respectively, at 100% breakthrough. The utilization of the binding sites was impacted by the ligand format and ligand density, rather than limitation in pore size of adsorbent as previously suggested. Overall, we demonstrated that multimeric fusion scFvs can successfully be developed and used as high-capacity ligands in affinity adsorbents, enabling lean process design and alignment with process specifications.


Subject(s)
Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/metabolism , Adsorption , Animals , Binding Sites , CHO Cells , Chromatography, Affinity/methods , Cricetulus , Cross-Linking Reagents/metabolism , HEK293 Cells , Humans , Ligands , Porosity , Protein Multimerization , Sepharose/metabolism
9.
Am J Phys Anthropol ; 139(2): 269-73, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19278018

ABSTRACT

Almost all mammals have brown or darkly-pigmented eyes (irises), but among primates, there are some prominent blue-eyed exceptions. The blue eyes of some humans and lemurs are a striking example of convergent evolution of a rare phenotype on distant branches of the primate tree. Recent work on humans indicates that blue eye color is associated with, and likely caused by, a single nucleotide polymorphism (rs12913832) in an intron of the gene HERC2, which likely regulates expression of the neighboring pigmentation gene OCA2. This raises the immediate question of whether blue eyes in lemurs might have a similar genetic basis. We addressed this by sequencing the homologous genetic region in the blue-eyed black lemur (Eulemur macaco flavifrons; N = 4) and the closely-related black lemur (Eulemur macaco macaco; N = 4), which has brown eyes. We then compared a 166-bp segment corresponding to and flanking the human eye-color-associated region in these lemurs, as well as other primates (human, chimpanzee, orangutan, macaque, ring-tailed lemur, mouse lemur). Aligned sequences indicated that this region is strongly conserved in both Eulemur macaco subspecies as well as the other primates (except blue-eyed humans). Therefore, it is unlikely that this regulatory segment plays a major role in eye color differences among lemurs as it does in humans. Although convergent phenotypes can sometimes come about via the same or similar genetic changes occurring independently, this does not seem to be the case here, as we have shown that the genetic basis of blue eyes in lemurs differs from that of humans.


Subject(s)
Eye Color/genetics , Lemuridae/genetics , Phenotype , Animals , Base Sequence , DNA Primers/genetics , Guanine Nucleotide Exchange Factors/genetics , Humans , Membrane Transport Proteins/genetics , Molecular Sequence Data , Polymorphism, Single Nucleotide/genetics , Sequence Alignment , Sequence Analysis, DNA , Species Specificity , Ubiquitin-Protein Ligases
11.
Eng Life Sci ; 19(7): 490-501, 2019 Jul.
Article in English | MEDLINE | ID: mdl-32625026

ABSTRACT

Single-chain variable fragments (scFv) are widely used in several fields. However, they can be challenging to purify unless using expensive Protein L-based affinity adsorbents or affinity tags. In this work, a purification process for a scFv using mixed-mode (MM) chromatography was developed by design of experiments (DoE) and proteomics for host cell protein (HCP) quantification. Capture of scFv from human embryonic kidney 293 (HEK293) cell feedstocks was performed by hydrophobic charge induction chromatography (MEP HyperCel™), whereafter polishing was performed by anion hydrophobic MM chromatography (Capto Adhere™). The DoE designs of the polishing step included both binding and flow-through modes, the latter being the standard mode for HCP removal. Chromatography with Capto Adhere™ in binding-mode with elution by linear salt gradient at pH 7.5 resulted in optimal yield, purity and HCP reduction factor of 98.9 > 98.5%, and 14, respectively. Totally, 258 different HCPs were removed, corresponding to 84% of identified HCPs. The optimized conditions enabled binding of the scFv to Capto Adhere™ below its theoretical pI, while the majority of HCPs were in the flow-through. Surface property maps indicated the presence of hydrophobic patches in close proximity to negatively charged patches that could potentially play a role in this unique selectivity.

12.
Neurosci Lett ; 428(1): 47-51, 2007 Nov 20.
Article in English | MEDLINE | ID: mdl-17945418

ABSTRACT

Although loss of cholinergic neurons in the basal forebrain is considered a key initial feature in Alzheimer's disease (AD), changes in other transmitter systems, including serotonin and 5-HT(2A) receptors, are also associated with early AD. The aim of this study was to investigate whether elimination of the cholinergic neurons in the basal forebrain directly affects 5-HT(2A) receptor levels. For this purpose intraventricular injection of the selective immunotoxin 192 IgG-Saporin was given to rats in doses of either 2.5 or 5 microg. The rats were sacrificed after 1, 2, 4 and 20 weeks. 5-HT(2A) protein levels were determined by western techniques in frontal cortex and hippocampus. A significant 70% downregulation in frontal cortex and a 100% upregulation in hippocampus of 5-HT(2A) receptor levels were observed 20 weeks after the cholinergic lesion when using the highest dose of 192 IgG-Saporin. Our results show that cholinergic deafferentation leads to decreased frontal cortex and increased hippocampal 5-HT(2A) receptor levels. This is probably a consequence of the interaction between the serotonergic and the cholinergic system that may vary depending on the brain region.


Subject(s)
Acetylcholine/metabolism , Brain Injuries/pathology , Frontal Lobe/metabolism , Hippocampus/metabolism , Neurons/pathology , Receptor, Serotonin, 5-HT2A/metabolism , Analysis of Variance , Animals , Antibodies, Monoclonal , Brain Injuries/chemically induced , Dose-Response Relationship, Drug , Male , Neurons/drug effects , Neurons/metabolism , Rats , Rats, Wistar , Ribosome Inactivating Proteins, Type 1 , Saporins , Time Factors
13.
J Chromatogr A ; 1468: 143-153, 2016 Oct 14.
Article in English | MEDLINE | ID: mdl-27663728

ABSTRACT

Monoclonal antibody (mAb) based affinity resins usually suffer from low binding capacity, most probably as a result of steric hindrance by the large 150kDa size of the mAb and a random immobilisation approach. The present work investigates the influence of a variety of factors on dynamic binding capacity (DBC) such as pore/ligand size ratio, accessibility of ligand and ligand density. The effect of pore/ligand size ratio was investigated using Fab and scFv fragments on various resins with different pore sizes. The accessibility of the ligand was investigated by a site-directed immobilisation approach, where three C-terminal tags, PPKPPK, FLAG™ and Cys, were introduced into the Fab fragments for immobilisation on resins via amino-, carboxyl- and thiol-groups, respectively. The scFv fragments were tagged at the C-terminal only with FLAG™ to enable a straight forward purification procedure, and were immobilised to resins via amino- and carboxyl-groups. The target protein had a molecular weight (MW) of 50kDa. A 3-fold higher dynamic binding capacity at 100% breakthrough (DBC100%) was observed for Fab wild-type (wt) on CNBr-activated Sepharose 4 FF relative to mAb on same resin at the same ligand density. However, no major difference in DBC100% was observed between Fab wt and scFv immobilised on CNBr-activated Sepharose 4 FF at the same ligand density. Thus, further increase of pore/ligand size ratio from Fab to scFv on a resin with average pore size of 300Å, did not seem to be beneficial. Among the tested tags, only the C-terminal Cys tag proved to site-direct the ligands during immobilisation as it allowed the DBC100% to increase 1.6-fold as compared to Fab wt immobilised via amino-groups on CNBr-activated Sepharose 4 FF and Actigel ALD Superflow at the same ligand density. The influence of ligand density was investigated by selecting immobilised Fab Cys on Sulfhydryl-reactive resin. Increasing ligand density from 0.103 to 0.202µmol/mL resulted in the same utilisation yield (82-85%), whereas a further increase in ligand density from 0.202 to 0.328µmol/mL resulted in a 20%-unit decrease in utilisation yield and less steep breakthrough curve, suggesting steric hindrance in the pores of the resin. In addition, site-directed affinity ligands resulted in a more pronounced, sigmoid-shaped breakthrough curve, leading to more efficient use of capacity. The highest DBC100% was obtained for Fab Cys on Sulfhydryl-reactive resin and scFv on Actigel ALD Superflow; 11mg/mL and 10mg/mL, respectively, as compared to the DBC100% of 0.8mg/mL for mAb on CNBr-activated Sepharose 4 FF. Pore/ligand size ratio of 3, which was achieved for Fab ligands on the studied resins, was shown to be feasible for capturing a protein in MW of 50kDa. Totally, a 13.8-fold improvement in DBC100% was achieved with the Fab-based affinity resin coupled via the C-terminal Cys as compared to the mAb-based affinity resin.


Subject(s)
Anion Exchange Resins/chemistry , Antibodies, Monoclonal/chemistry , Chemistry Techniques, Analytical/methods , Ligands , Sepharose/analogs & derivatives , Sulfhydryl Compounds/chemistry , Anion Exchange Resins/metabolism , Antibodies, Monoclonal/metabolism , Particle Size , Sepharose/chemistry , Single-Chain Antibodies/metabolism
14.
Acta Neuropathol Commun ; 4: 40, 2016 Apr 22.
Article in English | MEDLINE | ID: mdl-27101868

ABSTRACT

Primary central nervous system lymphoma (PCNSL) is an aggressive variant of diffuse large B-cell lymphoma (DLBCL) confined to the CNS. TP53 mutations (MUT-TP53) were investigated in the context of MIR34A/B/C- and DAPK promoter methylation status, and associated with clinical outcomes in PCNSL patients. In a total of 107 PCNSL patients clinical data were recorded, histopathology reassessed, and genetic and epigenetic aberrations of the p53-miR34-DAPK network studied. TP53 mutational status (exon 5-8), with structural classification of single nucleotide variations according to the IARC-TP53-Database, methylation status of MIR34A/B/C and DAPK, and p53-protein expression were assessed. The 57/107 (53.2 %) patients that were treated with combination chemotherapy +/- rituximab (CCT-treated) had a significantly better median overall survival (OS) (31.3 months) than patients treated with other regimens (high-dose methotrexate/whole brain radiation therapy, 6.0 months, or no therapy, 0.83 months), P < 0.0001. TP53 mutations were identified in 32/86 (37.2 %), among which 12 patients had hotspot/direct DNA contact mutations. CCT-treated patients with PCNSL harboring a hotspot/direct DNA contact MUT-TP53 (n = 9) had a significantly worse OS and progression free survival (PFS) compared to patients with non-hotspot/non-direct DNA contact MUT-TP53 or wild-type TP53 (median PFS 4.6 versus 18.2 or 45.7 months), P = 0.041 and P = 0.00076, respectively. Multivariate Cox regression analysis confirmed that hotspot/direct DNA contact MUT-TP53 was predictive of poor outcome in CCT-treated PCNSL patients, P = 0.012 and P = 0.008; HR: 1.86 and 1.95, for OS and PFS, respectively. MIR34A, MIR34B/C, and DAPK promoter methylation were detected in 53/93 (57.0 %), 80/84 (95.2 %), and 70/75 (93.3 %) of the PCNSL patients with no influence on survival. Combined MUT-TP53 and MIR34A methylation was associated with poor PFS (median 6.4 versus 38.0 months), P = 0.0070. This study suggests that disruption of the p53-pathway by MUT-TP53in hotspot/direct DNA contact codons is predictive of outcome in CCT-treated PCNSL patients, and concomitant MUT-TP53 and MIR34A methylation are associated with poor PFS.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Central Nervous System Neoplasms , Lymphoma , Mutation/genetics , Pharmacogenetics , Tumor Suppressor Protein p53/genetics , Analysis of Variance , Antigens, CD/metabolism , Central Nervous System Neoplasms/drug therapy , Central Nervous System Neoplasms/genetics , Central Nervous System Neoplasms/mortality , DNA Methylation/genetics , DNA Mutational Analysis , Death-Associated Protein Kinases/genetics , Death-Associated Protein Kinases/metabolism , Denmark , Female , Humans , Lymphoma/drug therapy , Lymphoma/genetics , Lymphoma/mortality , Male , MicroRNAs/genetics , MicroRNAs/metabolism , Predictive Value of Tests , Retrospective Studies , Survival Analysis , Treatment Outcome
15.
Biochem J ; 378(Pt 2): 421-33, 2004 Mar 01.
Article in English | MEDLINE | ID: mdl-14572311

ABSTRACT

Previous enzyme kinetic and structural studies have revealed a critical role for Asp181 (PTP1B numbering) in PTP (protein-tyrosine phosphatase)-mediated catalysis. In the E-P (phosphoenzyme) formation step, Asp181 functions as a general acid, while in the E-P hydrolysis step it acts as a general base. Most of our understanding of the role of Asp181 is derived from studies with the Yersinia PTP and the mammalian PTP1B, and to some extent also TC (T-cell)-PTP and the related PTPa and PTPe. The neighbouring residue 182 is a phenylalanine in these four mammalian enzymes and a glutamine in Yersinia PTP. Surprisingly, little attention has been paid to the fact that this residue is a histidine in most other mammalian PTPs. Using a reciprocal single-point mutational approach with introduction of His182 in PTP1B and Phe182 in PTPH1, we demonstrate here that His182-PTPs, in comparison with Phe182-PTPs, have significantly decreased kcat values, and to a lesser degree, decreased kcat/Km values. Combined enzyme kinetic, X-ray crystallographic and molecular dynamics studies indicate that the effect of His182 is due to interactions with Asp181 and with Gln262. We conclude that residue 182 can modulate the functionality of both Asp181 and Gln262 and therefore affect the E-P hydrolysis step of PTP-mediated catalysis.


Subject(s)
Models, Chemical , Protein Tyrosine Phosphatases/chemistry , Protein Tyrosine Phosphatases/metabolism , Amino Acid Sequence , Amino Acids/chemistry , Aspartic Acid/chemistry , Catalysis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Histidine/chemistry , Humans , Hydrolysis , Models, Molecular , Mutagenesis, Site-Directed , Nitrophenols/metabolism , Organophosphorus Compounds/metabolism , Peptides/chemistry , Peptides/metabolism , Phenylalanine/chemistry , Phenylalanine/genetics , Phosphotyrosine/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatase, Non-Receptor Type 3 , Protein Tyrosine Phosphatases/genetics , Sequence Alignment , Vanadates/chemistry
16.
Article in English | MEDLINE | ID: mdl-14659430

ABSTRACT

Our structure-based drug discovery program within the field of protein-tyrosine phosphatases (PTPs) demands delivery of significant amounts of protein with extraordinary purity specifications over prolonged time periods. Hence, replacement of classical, multi-step, low-yield protein purifications with efficient affinity techniques would be desirable. For this purpose, the highly selective PTP1B inhibitor 2-(oxalyl-amino)-4,5,6,7-tetrahydro-thieno[2,3-c]pyridine-3-carboxylic acid (OTP) was coupled to epoxy-activated Sepharose 6B (OTP Sepharose) and used for one-step affinity purification of tag-free PTP1B. The elution was performed with a combined pH and salt gradient. Importantly, since OTP Sepharose binds PTP1B with an intact active site only, the method ensures that the purified enzyme is fully active, a feature that might be particularly important in PTP research.


Subject(s)
Chromatography, Affinity/methods , Enzyme Inhibitors/pharmacology , Protein Tyrosine Phosphatases/isolation & purification , Catalytic Domain , Chromatography, Gel , Cloning, Molecular , DNA, Complementary , Hydrogen-Ion Concentration , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatases/antagonists & inhibitors , Protein Tyrosine Phosphatases/chemistry , Protein Tyrosine Phosphatases/genetics , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification
17.
J Biotechnol ; 173: 76-85, 2014 Mar 10.
Article in English | MEDLINE | ID: mdl-24445170

ABSTRACT

Site selective chemical modification is a preferred method, employed to prolong the circulation half-life of biopharmaceuticals. Cysteines have been used as attachment point for such modification, however, to be susceptible for chemical modification the involved thiol must be in its reduced form. Proteins often contain disulfides, which aid to maintain their tertiary structure and therefore must remain intact. Thus, methods for selectively reducing cysteine residues, introduced through site-directed mutagenesis, are of interest. In this study a macroporous, polymeric monolith was designed for selectively reducing a single cysteine residue inserted in recombinant human growth hormone (hGH). Advantages of such a material are the circumvention of the need to remove the reducing agent after reaction, as well as milder reduction conditions and a concomitant lower risk of reducing the native disulfides. The designed monolith showed very high capacity towards the selective reduction of an unpaired cysteine residue in a recombinant hGH variant. Factors influencing the selectivity and rate of reaction were investigated and it was found that monolith thiol loading, and buffer pH had an effect on the rate of reduction, whereas hGH variant concentration and buffer conductivity influenced both rate of reduction and selectivity. The developed system constitutes the basis for the development of a scalable platform for selective reduction of a capped cysteine residue in hGH.


Subject(s)
Cryogels/chemistry , Cysteine/metabolism , Disulfides/metabolism , Human Growth Hormone/metabolism , Sulfhydryl Compounds/chemistry , Half-Life , Humans , Microscopy, Electron, Scanning , Models, Chemical , Mutagenesis, Site-Directed , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism
18.
Oncotarget ; 5(7): 1912-25, 2014 Apr 15.
Article in English | MEDLINE | ID: mdl-24722400

ABSTRACT

MiR34A, B and C have been implicated in lymphomagenesis, but information on their role in normal CD19+ B-cells (PBL-B) and de novo diffuse large B-cell lymphoma (DLBCL) is limited. We show that in normal and activated B-cells miR34A-5p plays a dominant role compared to other miR34 family members. Only miR34A-5p is expressed in PBL-B, and significantly induced in activated B-cells and reactive lymph nodes. In PBL-B, the MIR34A and MIR34B/C promoters are unmethylated, but the latter shows enrichment for the H3K4me3/H3K27me3 silencing mark. Nine de novo DLBCL cases (n=150) carry both TP53 mutation and MIR34A methylation ("double hit") and these patients have an exceedingly poor prognosis with a median survival of 9.4 months (P<0.0001), while neither TP53 mutation, MIR34A or MIR34B/C promoter methylation alone ("single hit") influence on survival. The TP53/MIR34A "double-hit" is an independent negative prognostic factor for survival (P=0.0002). In 2 DLBCL-cell lines with both TP53 mutation and promoter methylation of MIR34A, miR34A-5p is upregulated by 5-aza-2'deoxycytidine. Thus, the TP53/MIR34A "double hit" characterizes a very aggressive subgroup of DLBCL, which may be treatable with epigenetic therapy prior to or in combination with conventional immunochemotherapy.


Subject(s)
Lymphoma, Large B-Cell, Diffuse/genetics , MicroRNAs/genetics , MicroRNAs/metabolism , Promoter Regions, Genetic/genetics , Tumor Suppressor Protein p53/genetics , Aged , Antigens, CD19/analysis , B-Lymphocytes/chemistry , B-Lymphocytes/metabolism , Cell Line, Tumor , Codon, Nonsense , Female , Humans , Lymph Nodes/metabolism , Lymphoma, Large B-Cell, Diffuse/drug therapy , Lymphoma, Large B-Cell, Diffuse/metabolism , Male , Methylation , Middle Aged , Mutation, Missense , Prognosis , Survival Rate
19.
Ugeskr Laeger ; 169(10): 925-7, 2007 Mar 05.
Article in Danish | MEDLINE | ID: mdl-17359739

ABSTRACT

On the basis of two case reports, this paper gives an overview of hepatitis E with respect to clinical features and occurrence in a non-endemic country like Denmark. Both reported cases were imported from endemic countries. The article includes a brief overview of the recent virology, epidemiology and clinical features of hepatitis E, with emphasis on the most frequently imported cases in Denmark.


Subject(s)
Hepatitis E , Denmark/epidemiology , Denmark/ethnology , Diagnosis, Differential , Female , Hepatitis E/diagnosis , Hepatitis E/drug therapy , Hepatitis E/epidemiology , Humans , Male , Travel
20.
Bioorg Med Chem ; 15(13): 4382-95, 2007 Jul 01.
Article in English | MEDLINE | ID: mdl-17482822

ABSTRACT

A two-step strategy was used for the preparation of C-terminally PEGylated hGH-derivatives. In a first step a CPY-catalyzed transpeptidation was performed on hGH-Leu-Ala, introducing reaction handles, which were used in the second step for the ligation of PEG-moieties. Both oxime-ligation and copper(I) catalyzed [2+3]-cycloaddition reactions were used for the attachment of PEG-moieties. The biological data show a dependency of the potency of the hGH-derivatives on both size as well as shape of the PEG-group.


Subject(s)
Human Growth Hormone/analogs & derivatives , Human Growth Hormone/chemistry , Polyethylene Glycols/chemistry , Capillary Electrochromatography , Chromatography, High Pressure Liquid , Human Growth Hormone/pharmacology , Humans , Indicators and Reagents , Polyethylene Glycols/pharmacology , Receptors, Somatotropin/drug effects , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Spectrophotometry, Ultraviolet
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